α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFβ antibody to promote durable rejection and immunity in squamous cell carcinomas

Abstract

Background: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFβ signaling.

Methods: To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFβ or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses.

Results: We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFβ monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFβ, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFβ antibody administration attenuates these effects. We show that α-TGFβ acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFβ acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery.

Conclusions: We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFβ-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFβ combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFβ/α-PD-1 combination therapy (NCT02947165).

Keywords: Checkpoint blockade; Epithelial mesenchymal transition (EMT); Squamous cell carcinoma; Tregs; Tumor mutation load; pSmad signaling; α-TGFβ /α-PD-1 combinatorial immunotherapy.

Conflict of interest statement

Ethics approval and consent to participate

All animal procedures adhered to NIH Guidelines for the Care and Use of Laboratory Animals and were undertaken under authorization of the UCSF Institutional Animal Care and Use Committee in an AAALAC approved facility.

Consent for publication

Not applicable

Competing interests

RJA, AM and OL are co-inventors of pending US patent 10167334 co-owned by UCSF and Xoma RJA and DS receive funding through a UCSF collaboration with Pfizer/CTI. DS owns stock in Pliant Therapeutics and has received more than $10,000 in consulting income from Pliant Therapeutics.. RJA has a Sponsored Research Agreement with Plexxikon Inc. DS has Sponsored Research Agreements with Pliant Therapeutics and Abbvie and is a co-inventor of 12 awarded patents and 6 pending patents owned or co-owned by the University of California, San Francisco. AB received funding through a collaboration with Bayer. AB is on the Advisory Boards of Mission Bio and InteRNA. MK owns stock in Pionyr Immunotherapeutics, and receives funding from Amgen, BMS and Abbvie to support the UCSF Immunoprofiler project. AM is currently an employee of Gilead and has direct equity ownership in XOMA Corporation and Gilead Sciences Inc. AM and OL were full time employees of XOMA Corporation during the period that this work was executed. AM and OL are coinventors on the following patents: Antibodies that bind Interleukin 2 and uses thereof – 2016 U.S. Provisional Utility Application M. Roell; A. Mirza; et al. Treatment of Cancer Using Inhibitors of TGFbeta and PD-1 – 2015. WIPO Patent Application WO/2016/161410A2 U.S. Prov. Utility Appl. No. 62/143,016; A. Mirza; O. Li; R. Akhurst. Antibodies targeting PTH1R to affect Humoral Hypercalemia of Malignancy and cancer – 2016 U.S. Provisional Utility Application A. Mirza; R. Levy; T. Takeuchi; D. Bedinger; and R. Hunt. Antibodies Specific for TGF-BETA – 2013 US 8,569,462; D. Bedinger; S. Khan; A. Mirza; A. Narasimha; T. Takeuchi. PRLR-Specific Antibody and Uses Thereof – 2008 WIPO Patent Application WO WO/2008/022295; D. Bedinger; J. Damiano; M. Luqman; L. Masat; A. Mirza; G. Nonet. Uses of Anti-CD40 Antibodies – 2008. WIPO Patent Application WO/2009/062054; M. Luqman; Y. Wang; S. Kantak; S. Hsu; A. Mirza. siRNA Libraries – 2004 WIPO Patent Application WO/2004/108897; C. Beraud; A. Mirza. OL is currently an employee of Five Prime Therapeutics, and has more than $10,000 in stock she is an inventor on three patents: including two listed above and Antibody fragments against the insulin receptor and uses thereof to treat hypoglycemia. DSM is an employee of Idorsia Pharmaceuticals Ltd. and holds equity in that company. MDT is an employee of, and holds ownership of equity in, Northern Biologics. MB is a coinventor on patent application PCT/US2015/052682, Modulation of stimulatory and non-stimulatory myeloid cells. MLB is currently an employee of Bristol-Myers Squibb.

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Figures

Fig. 1

α-PD-1 effects on tumor-growth of chemically-induced and GEMM-derived SCCs. a Total NS-SNV loads of Kras-driven SCCs, determined by WES analysis. The panel includes four chemically-induced (CCK) and two GEMM-derived (GEK) SCCs, b Scheme for syngeneic tumor generation and drug therapy: After implantation of 1.5 × 104 tumor cells by unilateral dorsolateral subcutaneous injection, tumors grew for 14 days until they reached ≥5 mm diameter. Mice were then treated with control IgGs or α-PD-1 drugs injected intraperitoneally (ip) into the contralateral side, with three drug administrations, each four days apart (considered days 0, 4 and 8). c-f) Tumor growth of the indicated tumor lines with or without α-PD-1 therapy was measured at least every other day from the time of first drug administration. Red arrows indicate timing of drug administration (see Additional file 3: Figure S1 for GEK1428, and CCK62 growth curves), c-e) mean tumor growth of 7–10 mice per arm. ** = p < 0.01 (Fisher’s Exact test)

Fig. 2

Alteration in tumor infiltrating leukocyte profiles in response to α-PD-1 therapy. Tumors, generated according to the scheme in Fig. 1b, were harvested 7 days after the first drug treatment and analyzed by (a) immunohistochemistry or (b-g) 11-color flow cytometry. a Immunohistochemical staining shows CD3+ total T cells, CD8a + cytotoxic T cells and CD45+ immune cells in the non-responsive tumor line, CCK62, and in the responsive tumor line, CCK168. b-g Flow cytometric analysis of CCK168 immune cell subsets in response to α-PD-1 therapy: b CD11b + Gr1-Ly6C-Ly6Clo myeloid cells, c total CD4+ T cells, d cytotoxic CD8+ T cells, e CD4 + CD25 + Foxp3+ Treg cells, f CD4 + Foxp3-CD25- Th cells, g ratio of CD4+ Th/Treg cells. Cell numbers shown are relative to 100, 000 CD45+ immune cells. Representative data of ≥ three biological replicates. Scale bars in (a) represents 50 μM in upper panels and 200 μM in lower panel. * = p < 0.05; ** = p < 0.01; *** = p < 0.001: Mann Whitney U test

Fig. 3

α-TGFβ and α-PD-1 synergize in eliciting tumor rejection in a CD8a + cell-dependent manner. a Growth curves of each of the six SCC lines, as indicated, after treatment with control IgG or α-TGFβ/α-PD-1 combination therapy on days 0, 4 and 8 (n = 8–10 mice per arm). Tumors were measured every other day after tumor implantation and therapy (see Fig. 1b). Summary of tumor responses for CCK168 (b) and CCK169 (d), and overall survival for CCK168 (c) following treatment on days 0, 4 and 8 with control IgG, α-PD-1, α-TGFβ or combination therapy. In (b) and (d), individual tumors were classified as complete responders (CR), partial responders (PR), or progressive disease (PD), according to their growth characteristics (CR, tumor eradicated with no regrowth; PR, tumor shrinkage ≥30%; PD, no effect of drugs compared to control IgG). For each drug arm, the percentages of total tumors within each response group are shown. n = 10 mice per arm per experiment, n = 3 independent experiments. c Kaplan Meier survival plot for mice bearing CCK168 tumors, after treatment on days 0, 4 and 8 with the indicated drugs. 2000 mm3 tumor size was used as the cut off for survival. e Average CCK168 tumor growth curves following treatment with α-PD-1/α-TGFβ combination therapy versus isotype control IgGs in mice with or without CD8+ T cell depletion. Animals were treated as in Fig. 1b, except that 24 h prior to the first therapeutic drug dose, mice received ip injection of CD8a + cell-depleting antibody or isotype matched control IgG. f) CCK168 tumor-bearing mice were administered one of six IgG combinations, either human α-pan-TGFβ IgG2 (XPA.42.068), human α-TGFβ1/β2 IgG2 (XPA.42.089) or human α-keyhole limpet hemocyanin as control IgG2, together with rat α-PD-1 IgG2 or rat IgG2 control, on days 0, 4 and 8. Average tumor volume (mm3) +/− SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001: Fisher’s exact test (b), Gehan-Breslow-Wilcoxon test (c), Student’s test (e, f)

Fig. 4

Immunophenotyping of CCK168 tumors in response to α-PD-1 and α-TGFβ therapy. a-k) CCK168 tumor cells were implanted sc into FVB mice according to Fig. 1b. After two drug doses on day 0 and 4, when some tumors began to show evidence of shrinkage, all tumors were harvested and analyzed by (a) immunohistochemistry or (b-k) multicolor flow cytometry. a Representative images of CD8a and CD45 immunohistochemistry for tumors from each of the four drug arms. Six to seven tumors were analyzed per drug arm per stain. Scale bar represents 50 uM. (b-k) Flow cytometry analysis shows increases in b CD45+ cells per live tumor cell, c CD8+ cytotoxic T cells per CD45+ immune cells and d increases in percentage of ICOS+ expressing CD8+ T cells. e α-PD-1 or α-TGFβ monotherapy elevates total CD4+ T cells with no additive effect. f α-TGFβ monotherapy neutralizes α-PD-1 induction of Tregs and, in combination therapy, reduces Treg levels to below baseline. g heterogeneous increase in CD4 + Th/CD4 + Treg ratio by α-TGFβ, h synergistic induction of CD8+/Treg ratios by α-PD-1 and α-TGFβ. The latter increased six to 40 fold in response to combinatorial therapy. (i) MHCII+CD11b + and (j) MHCII+CD11c + myeloid cells diminish as a percentage of total CD45 + cells following α-PD-1 or α-TGFβ therapy. k The ratio of mature T cells (CD4+ plus CD8+ cells) per CD11b myeloid cell (CD45 + Ly6G-CD11b + MHCII+) increases after combinatorial therapy. Flow cytometry data are representative of two to seven independent experiments for each cell type. * p < 0.05; ** p < 0.01; *** p < 0.001: Mann Whitney U test

Fig. 5

Treg depletion synergizes with α-PD-1 but not α-TGFβ in tumor regression and long term survival. CCK168 tumors were treated as in Fig. 1b, except that 24 h prior to the first therapeutic drug dose, mice received intraperitoneal injection of an IgG control or α-CD25+ cell-depleting antibody. a) and b) Kaplan Meier survival plots for CCK168 tumor-bearing mice using using 2000 mm3 tumor size as cut off for survival. P < 0.05 = *, p < 0.01 = ** Gehan-Breslow-Wilcoxon test

Fig. 6

α-PD-1 therapy induces pSmad3 signaling in CCK168 tumors to enhance EMT and suppress gene expression of antigen presenting machinery. a-f CCK168 tumor-bearing mice were treated either with α-PD-1 monotherapy or IgG control antibodies on day 0 and 4. As indicated in (a), on day 8, the α-PD-1 treated group were randomly split into two further groups and treated with either control IgG or α-TGFβ monotherapy, and all tumors were harvested 2 h later for pSmad3 immunofluorescence staining. b and c quantification of the three arms of the experiment b as percentage of DAPI+ nuclei stained with pSmad3 and c intensity of pSmad3 staining per nucleus. d-f representative images of pSmad3 immunofluorescence staining. Note rim of tumor in α-TGFβ treated sample f shows dramatically reduced pSmad3 staining. g, h CCK168 tumor-bearing mice were treated with α-PD-1 or α-TGFβ on day 0 and day 4, and analyzed by pSmad3 immunofluorescence on day 8. i-n CCK168 cells grown in vitro were treated with TGFβ and/or α-TGFβ antibodies. I-m) phase contrast analysis shows reversible TGFβ-induced EMT. n RNA from cultures shown in (l-m) was extracted and subjected to qRT-PCR to quantify gene expression of components of the tumor cell antigen presentation machinery, Mhc1, B2M (β2-microglobulin), Tap1 and Tap2. * = P < 0.05, **** = P < 0.0001; Unpaired two-tailed Student’s T test

Fig. 7

Heterogeneous mechanisms of tumor response to α-PD-1 and α-TGFβ therapy. a Unsupervised hierarchical clustering of indicated immune cell sub-populations in tumors after treatment with control IgG, α-PD-1, α-TGFβ, or combination therapy. Tumors were classified into responders (light green), stable disease (dark green) or non-responders (red) as described in Methods (colored horizontal bar). Responding mice tend to cluster into two groups that we label Responding Cluster A and Responding Cluster B, while non-responding mice tend to cluster into a third group (Cluster C). Mice in the two responding groups had significantly better outcomes than mice in the nonresponding cluster (p = 0.02, Fisher’s Exact test). Immune profiles of tumor infiltrates in Cluster B are characterized by high T cell levels and relatively low LyC6–macrophages (TAM2s), while those in Cluster A are characterized by low levels of all immune cell subtypes except CD4+ T cells, in particular high CD4 + FoxP3–CD25– (T helper) cells. b Unsupervised hierarchical cluster analysis undertaken according to expression of a Treg transcriptomic signature in pre-treatment human melanoma samples from patients treated with α-PD-1 (see Supplementary Methods). Transcriptomic data from pre-treatment melanoma samples [12] were subjected to unsupervised hierarchical cluster analysis based on gene transcripts whose expression correlates with FoxP3 expression in CD4+ cells. Human tumors responses were classified according to Hugo et al. 2016 [12] and the original tumor IDs are presented at the base of the figure. Samples from patients exhibiting a response, in particular CR, tended to cluster together (P = 0.005, Fisher’s Exact test) and to have reduced expression of FoxP3-associated genes. c,d) Each SCC line, in order of decreasing TML, CCK168, CCK169, CCK62, CCK166, GEK1425, GEK1428, was used to induced tumors in mice, and treated with α-PD-1 or control IgG on Day 0 and Day 4 (Fig. 1b). On day 8, tumors were harvested for flow cytometric analysis of immune cells. c CD4+ T cells per total CD45+, d CD8+ cytotoxic T cells per total CD45+